For many years we have been studying how these polyamines are synthesized, how their biosynthesis and degradation are regulated, their physiologic functions and how they act in vivo. For this purpose we have constructed null mutants in each of the biosynthetic steps in both Escherichia coli and Saccharomyces cerevisiae, and have prepared over-expression systems for the biosynthetic enzymes. As part of our studies on the biological functions of the polyamines we have used a chemostat system and our mutant of Escherichia coli that lacks all the genes for the polyamine biosynthesis for a global transcription analysis on the effect of added polyamines. The results showed that 80 genes were up-regulated more than 2-fold and 25 genes were up-regulated more than 3-fold within a short time after the polyamine addition. Polyamine addition also caused more than 2-fold down-regulation of 51 genes. Particularly striking was the 2- fold to 5-fold increase in the expression of many genes in the E. coli acid resistance (AR) pathway (gadA, gadB, gadC, gadX, gadY, slp, ybaS, hdeD). This finding is of particular interest because to colonize the mammalian gastrointestinal tract, E. coli must survive passage through the acidic environment of the stomach. In our most recent study we have focused our attention on the regulatory network responsible for polyamine induced glutamate decarboxylase activation. Since we found that the strain used for the above microarray experiment contained an amber mutation in the gene coding for alternate sigma factor for RNA polymerase (rpoS) at codon 33 (TAG instead of CAG) we changed the strain by successive P1 transductions to convert it from rpoS(amb) to rpoS(WT). We then used this new strain for another set of microarrays. We found that within 60 minutes after polyamine addition 54 genes were up-regulated more than 2-fold and 15 genes were up-regulated more than 3-fold; 94 genes were down-regulated more than 2-fold. We confirmed our earlier studies (above) that the most striking early response to the polyamine addition is the increased expression of the genes for the glutamate dependent acid resistance system (GDAR). Not only were the two genes for glutamate decarboxylases (gadA and gadB) and the gene for glutamate gamma-aminobutyrate antiporter (gadC) induced by the polyamine addition, but also the various genes involved in the regulation of this system were induced. We confirmed the importance of polyamines for the induction of the proteins of the GDAR system by direct measurement of glutamate decarboxylase protein and activity and by measuring acid-survival at pH 2.5. In the absence of added polyamines there was no induction of glutamate decarboxylase and no protection against acid stress. The protection from acid stress required polyamines, glutamic acid, and glutamate decarboxylase (either GadA or GadB). The effect of deletions of the regulatory genes on the GDAR system and the effect of overproduction of three of these genes was also studied. Deletion of either rpoS (alternate Sigma factor) or gadE (a LuxR like regulator of GDAR pathway) or rcsB (a response regulator essential for binding to gadE), resulted in complete loss of glutamate decarboxylase activity and acid resistance in cultures grown in minimal medium even in the presence of polyamines. On the other hand, deletions of other regulatory genes, such as gadX or gadY showed modest effect on GAD induction by polyamines. Although there is a report in the literature that inhibition of cyclic AMP synthesis is the primary mechanism for the effect of polyamine addition on gad gene expression, in marked contrast to this report we have found that polyamine addition to the polyamine deficient mutants caused an increase in the cyclic AMP levels. We also found that even if the polyamine deficient strain contained a deletion of cyaA (adenylate cyclase) or crp (cAMP receptor prrotein), polyamine addition still resulted in a marked increase in glutamate decarboxylase induction and acid resistance at pH 2.5. Most strikingly, overproduction of the alternate sigma factor rpoS and of the regulatory gene gadE resulted in very high levels of glutamate decarboxylase and almost complete protection against acid stress even in the absence of any polyamines. A dose response study showed that at least 100 micromolar putrescine or 10 micromolar spermidine is needed to induce GAD activity in E. coli; cadaverine had a very small effect on GAD induction. As spermidine is most effective in inducing the enzyme activity and as we know from our earlier studies on glutathionylspermidine that in the stationary phase most of the free spermidine is converted to glutathionylspermidine in E. coli, we have inserted a gss (glutathionylspermidine synthetase-amidase) mutation into our polyamine mutant and showed that spermidine conjugated to glutathionylspermidine is more effective in inducing glutamate decarboxylase activity in the polyamine-deficient mutant than free spermidine. In conclusion, these data show that a major function of polyamines in E. coli is protection against acid stress by increasing the synthesis of glutamate decarboxylase, presumably by increasing the levels of the rpoS and gadE regulators. In a different but related study, we have developed a new polyamine deficient strain using the MG1655 E. coli strain as a parental organism by systematic deletions of the genes encoding the enzymes for the polyamine biosynthetic pathway. This is important as the E. coli polyamine mutant we have been using has a long history with many exposures to mutagenic agents and therefore a comparable wild type strain is lacking for any detailed genetic comparison. This new strain has similar phenotype and polyamine requirements as our old strain. This strain also shows acid sensitivity and absence of glutamate decarboxylase induction in the absence of polyamines and correction by added polyamines. Using this new strain we have recently performed a new set of microarray analyses. 130 genes are up-regulated more than 3-fold by polyamines and 90 genes are down-regulated more than 3-fold by polyamine supplementation. In addition to the genes in the GDAR system, many genes in the E. coli bio-film development and nitrate metabolism are induced after polyamine addition. The down-regulated genes fall into polyamine transport, molybdate transport and flagella development categories. One interesting genes that is up-regulated 4-fold by polyamines is dps, which encodes a stress response protein. As we have shown that polyamines modulate various stress responses (oxidative, acidic, etc), we plan to extend our studies to find out the pathway involved in polyamine induced regulation of dps, and its role in E. coli stress responses. Another gene hokA is repressed 4.8-fold by polyamine addition. hokA mRNA encodes a toxic transmembrane protein, which is know to maintain plasmid maintenance in E. coli. In another related study, we are now analyzing the same RNA samples used in the recent microarrays for RNA-seq analysis to obtain more complete information on polyamine induced gene regulation in E. coli.Data from RNA seq analyses between polyamine deficient and polyamine supplemented cultures showed a large increase in acid response genes as the primary effects of polyamine addition supporting the microarray data. Interestingly, we also found an antisense small RNA, which is involed in the regulation of gadE is to be upregulated by polyamine addition. As it is located in the intergenic region of gadE and hdeD genes this was not identified by microarrys. Further confirmation of the regulatory roles of rpoS and gadE are shown by a comparison of the genome wide expression profiling data from series of microarrays.